Solution Structure of MSL2 CXC Domain Reveals an Unusual Zn3Cys9 Cluster and Similarity to Pre-SET Domains of Histone Lysine Methyltransferases

Ye K (2012) Solution Structure of MSL2 CXC Domain Reveals an Unusual Zn3Cys9 Cluster and Similarity to Pre-SET Domains of Histone Lysine Methyltransferases. PLoS ONE 7(9): e45437. doi:10.1371/journal.pone.0045437 Solution Structure of MSL2 CXC Domain Reveals an Unusual Zn3Cys9 Cluster and Similarity to Pre-SET Domains of Histone Lysine Methyltransferases Sanduo Zheng 0 Jia Wang 0 Yingang Feng 0 Jinfeng Wang 0 Keqiong Ye 0 Michael Massiah, George Washington University, United States of America 0 1 Department of Biochemistry and Molecular Biology, College of Life Sciences, Beijing Normal University , Beijing , China , 2 National Institute of Biological Sciences , Beijing , China , 3 Shandong Provincial Key Laboratory of Energy Genetics, Qingdao Institute of BioEnergy and Bioprocess Technology, Chinese Academy of Sciences , Qingdao, Shangdong , China , 4 National Laboratory of Biomacromolecules, Institute of Biophysics, Chinese Academy of Sciences , Beijing , China The dosage compensation complex (DCC) binds to single X chromosomes in Drosophila males and increases the transcription level of X-linked genes by approximately twofold. Male-specific lethal 2 (MSL2) together with MSL1 mediates the initial recruitment of the DCC to high-affinity sites in the X chromosome. MSL2 contains a DNA-binding cysteine-rich CXC domain that is important for X targeting. In this study, we determined the solution structure of MSL2 CXC domain by NMR spectroscopy. We identified three zinc ions in the CXC domain and determined the metal-to-cysteine connectivities from 1H-113Cd correlation experiments. The structure reveals an unusual zinc-cysteine cluster composed of three zinc ions coordinated by six terminal and three bridging cysteines. The CXC domain exhibits unexpected structural homology to preSET motifs of histone lysine methyltransferases, expanding the distribution and structural diversity of the CXC domain superfamily. Our findings provide novel structural insight into the evolution and function of CXC domains. - Funding: K.Y. was supported by the National Basic Research Program of China (973 Program) (2010CB835402 and 2012CB910900) and Beijing Municipal Government. Y.F. was supported by the National Natural Science Foundation of China (30970571). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. . These authors contributed equally to this work. Organisms with different numbers of sex chromosomes between males and females face the problem of an unequal dosage of genes from sex chromosomes. In Drosophila melanogaster, the transcriptional level of most genes in the single male X chromosome is increased by approximately twofold to match that from two female X chromosomes (see recent reviews [13]). This dosage compensation process is mediated by the dosage compensation complex (DCC) or male-specific lethal (MSL) complex, which contains at least five proteins MSL1, MSL2, MSL3, males absent on the first (MOF) and maleless (MLE) and two non-coding RNAs roX1 and roX2. MSL1 is a scaffold protein associated with MSL2, MSL3 and MOF [46]. In male flies, the DCC is located at hundreds of sites along the length of the X chromosome. Each of five proteins and at least one of the functionally redundant roX RNAs are required for full association of the DCC on the X chromosome and for male viability. The DCC is not assembled in females because MSL2 translation is tightly repressed [7,8]. The DCC has been shown to primarily bind at bodies of active genes on the X chromosome [9,10]. The transcriptional activation is caused, at least in part, by the MOF-mediated acetylation of histone H4 lysine 16 and enhanced transcriptional elongation [11]. The mechanism by which the DCC is specifically localized to the X chromosome remains poorly understood. According to a prevalent model, the DCC first binds to a limited number of high-affinity sites (HAS) or chromatin entry sites (CES) in the X chromosome and then spreads in cis to flanking active genes [12]. The spreading process probably involves the interaction of the MSL3 chromodomain with trimethylated H3K36, a marker for actively transcribed genes [13]. HAS are able to attract even a partially assembled DCC that lacks MSL3, MOF or MLE, or a low concentration of DCC [7,14,15]. A body of evidence suggests that specific DNA sequences are involved in HAS recognition. When translocated to an autosome, HAS as short as 100200 base pairs (bp) can still recruit the DCC [1620]. Chromatin immunoprecipitation studies followed by microarray analysis or deep sequencing have mapped ,140 HAS on the X chromosome [20,21]. The binding sites of the DCC on HAS are enriched with a GA-rich MSL recognition element (MRE) [20]. However, the MRE motif occurs frequently outside of HAS and is only slightly enriched in the X chromosome over autosomes, indicating that the MRE motif is not the sole determinant for HAS recognition. The conformation of chromatin also appears to be important for HAS recognition, since HAS are characterized by low nucleosome occupancy [20] and special compartments in the nuclei [22]. MSL2 is a core component of the DCC [2325] and together with MSL1 is required for the DCC to bind HAS on the X [14,15,26,27]. MSL2 was recently shown to be a DNA-binding protein and specifically recognize a HAS in reporter gene assay in Drosophila cells [28]. However, MSL2 failed to discriminate the HAS sequence in vitro. An unknown selectivity cofactor was proposed to cooperate with MSL2 in vivo for specific HAS recognition [28]. MSL2 is composed of an N-terminal RING domain, a cysteinerich CXC domain and a C-terminal region rich in proline and basic residues (Pro/Bas patch). The RING domain binds MSL1 [6,29] and exhibits ubiquitin E3 ligase activity toward H2B K34 [30]. The CXC domain contributes critically to the DNA-binding activity of MSL2 [28]. CXC domains are also present, mostly in two copies, in the tesmin/TSO1 protein family [3135]. The tandem CXC domain of human LIN54 and soybean CPP1 has been shown to bind specific DNA sequences [32,33]. No structure has been reported for any CXC domain. The CXC domain is remarkable by having 9 invariant Cys within about 50 residue region. In this study, we have determined the first structure of MSL2 CXC domain by NMR spectroscopy. The structure reveals a compact fold that encages an unusual Zn3Cys9 cluster. Interestingly, the CXC structure with a Zn3Cys9 cluster shows strong similarity to pre-SET motifs of histone lysine methyltransferases, suggesting that the CXC and pre-SET domains share a common evolutionary origin. The MSL2 CXC Domain is an Autonomously Folded Structure Containing Three Zinc Ions Our structural analysis of D. melanogaster MSL2 CXC domain was conducted mainly on two constructs. One construct containing residues 517572 and the C560G mutation (CXC (...truncated)